Abstract Synapses, the structural units for transmitting electrochemical signals between neurons, form the basis of brain wiring and the specificity of synaptic connections determines brain function. During development, the nervous system acquires an excess of synapses that undergoes refinement, to optimize the signal-to-noise ratio. Developmental brain wiring is refined in part through synaptic pruning, which eliminates weak synapses allowing for strengthening of those retained. Synapse formation and elimination also persist in the mature nervous system through experience-dependent structural synaptic plasticity, which is the basis of learning. Therefore, synaptic formation and pruning are crucial not only in shaping neural circuits during development but also in regulating synaptic plasticity in response to experience and memory. Defects in synaptic pruning and maintenance have been implicated in neurodevelopmental disorders and neurodegeneration. Although synapses functionally connect neurons, the glial support cells such as microglia and astrocytes carry out the process of synapse pruning. For example, genetic manipulation of microglial complement and fractalkine receptor pathways in mice has conclusively demonstrated their involvement in synapse pruning. However, the full spectrum of molecular components involved in this process remains to be defined. GPR56 is a member of the adhesion-GPCR family. Our preliminary studies showed (1) cell-specifically deleting microglial Gpr56 in mice results in significantly increased synapses in a brain region called dorsal lateral geniculate ganglion (dLGN); (2) an isoform of Gpr56 (termed S4) is predominantly expressed in microglia and plays an indispensable function in microglia-mediated synaptic refinement; (3) GPR56 binds phosphatidylserine (PtdSer), a potential `tag' for synapse removal, in vitro; and (4) there are increased PtdSer+ synapses in dLGN upon deleting microglial Gpr56. Taken together, we hypothesize that the S4 variant of GPR56 regulates microglia-mediated developmental synapse refinement, possibly by binding to PtdSer. Our present proposal will test this hypothesis using genetic models and electrophysiological analysis, as well as biochemical and cellular assays. The success of the proposed research will establish a novel mechanism and signaling pathway in regulating developmental synapse refinement. We intend to lay the foundation to investigate the role(s) of microglial GPR56 in neurodevelopmental disorders as well as its potential role in neurodegeneration.